CN1463578A - Submerged culturing method for making mushroom liquid bacterial and culture medium therefor - Google Patents
Submerged culturing method for making mushroom liquid bacterial and culture medium therefor Download PDFInfo
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- CN1463578A CN1463578A CN 02124094 CN02124094A CN1463578A CN 1463578 A CN1463578 A CN 1463578A CN 02124094 CN02124094 CN 02124094 CN 02124094 A CN02124094 A CN 02124094A CN 1463578 A CN1463578 A CN 1463578A
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Abstract
The present invention provides one kind of deep fermentation process of culturing liquid Xianggu mushroom seed and its culture medium, and aims at providing one liquid Xianggu mushroom seed process superior to available technology. The culture process includes the steps of: mother seed culture in mother seed culturing medium; dark transitional culture of the obtained mycelium in sawdust culture medium at 22-27 deg.c for 12-20 days; the first stage of shaking culture of the crushed sawdust seed inside liquid culture medium for 5-7 days; the second stage of shaking amplification culture in fresh liquid culture medium; and fermentation treatment of the shaking cultured mushroom liquid to obtain liquid Xianggu mushroom seed product. The relevant culture medium includes culture medium for Xianggu mushroom mother seed, sawdust culture medium and deep fermenting culture medium.
Description
Technical field:
The present invention relates to the submerged culturing method for making and the medium thereof of strain fermentation cultural method, particularly mushroom liquid bacterial.
Background technology:
Mushroom production uses solid spawn to produce always, began to have the people in the research of carrying out the liquid fungus seed culture technique in recent years, but also there are a lot of problems in this bacterial classification that is used to produce, (1) female breeding of planting, the universal medium of many uses, be not specifically designed to the medium of mushroom strain, therefore female kind breeding time is longer, and mycelia is of low quality; (2) in the inoculation of producing liquid spawn, be mostly directly to carry out the one-level shake-flask culture with the bacterial classification that mother culture media is cultivated, time length, the bacterium ball of producing the bacterium ball like this are not of uniform size, are unfavorable for the growth of bacterium ball; (3) the used medium of liquid spawn contains compositions such as a large amount of glucose, sucrose, and economic cost is higher.
Summary of the invention:
The present invention overcomes the deficiency of existing mushroom liquid bacterial fermentation culture method, designs a kind of mushroom liquid bacterial submerged culturing method for making that can turn out the evenly moderate bacterial classification of size and shorten incubation time.
Another object of the present invention provides a kind of female special-purpose mother culture media of planting breeding that is used in the mushroom liquid bacterial incubation, this mother culture media can impel that mycelial growth is vigorous, growth time is short, mycelia is strong, mycelia quality height.
The 3rd purpose of the present invention provides a kind of wood chip pedigree seed culture medium that uses in the mushroom liquid bacterial incubation, this medium can provide growth vigorous, a uniform mycelia fragment of bacterium.
The 4th purpose of the present invention provides a kind of submerged fermentation culture medium that uses in the mushroom liquid bacterial incubation, this medium can reduce cost, and is beneficial to produce the high-quality bacterial classification.
Technical scheme of the present invention is:
A kind of cultural method of mushroom liquid bacterial: comprise the steps: 1, mother planted in mother culture media, cultivate; 2, will cultivate the mycelia culture transferring that covers with test tube in mother culture media and carry out transition in the wood chip pedigree seed culture medium and cultivate, condition of culture is: temperature 22-27 ± 1 ℃, secretly cultivated 12-20 days, and treat that mycelia covers with; 3, the wood chip bacterial classification is fully smashed after, culture transferring carries out the cultivation amplification that one-level is shaken bottle in the liquid nutrient medium immediately; 4, one-level being shaken in the bottle 5-7 days bacterium liquid of growth moves into the secondary fill the fresh liquid medium and shakes in the bottle and increase; 5, secondary being shaken the bacterium liquid that comes out of bottle carries out fermentation process and can obtain the finished product mushroom liquid bacterial.
Above-mentioned wood chip pedigree seed culture medium is the medium that contains wood chip.
Above-mentioned wood chip pedigree seed culture medium is the medium that contains wood chip, cotton seed hull and bean powder.
Above-mentioned wood chip pedigree seed culture medium is cultivated and is carried out in triangular flask.
The composition and the weight percent content scope of above-mentioned wood chip pedigree seed culture medium raw material part are: sucrose 0.5-1.5%, cotton seed hull 70-75%, wood chip 5-10%, wheat bran 5-10%, bean powder 2-6%, land plaster (CaCO
3) 0.5-2.5%, the wood chip pedigree seed culture medium by above-mentioned raw materials and water by 1: 1.2-1.4 mixes and gets, and its pH value is 5.5-6.5
Above-mentioned mother culture media is special-purpose mother culture media, in original potato culture, added mushroom waste material leachate, its pH value scope is 5.5-6.5, its composition and weight percent content scope are: potato 10-30%, mushroom waste material 1-5%, glucose 1-3%, yeast extract 0.02-2%, peptone 0.05-0.2%, KH
2PO
4: 0.05-0.2%, MgSO
47H
2O:0.01-0.07%, agar 1-3%, water: 60-85%.Condition of culture in the mother culture media is: temperature 20-27 ± 1 ℃, dark culturing, time 5-10 days.
The aforesaid liquid medium is a submerged fermentation culture medium, and its composition and weight percent content scope thereof are: yeast extract 0.1-0.5%, wheat bran 0.5-3%, corn flour 1-5%, starch 1-5%, land plaster (CaCO
3) 0.05-0.5%, KH
2PO
4: 0.02-0.1%, MgSO
47H
2O:0.01-0.2%, NaCl:0.01-0.1%, vitamin B1: 0.0001-0.008%, agar 0.05-0.9%, water: 86-96%.Adopt this submerged fermentation culture medium condition of culture of liquid spawn in I and II shakes bottle to be: temperature 25-27 ± 1 ℃, frequency of oscillation 125-155 rev/min, incubation time 3-5 days, inoculum concentration is different according to required product with the liquid nutrient medium coefficient, is respectively 10-15% and 100ml-150ml/500ml.Adopt this submerged fermentation culture medium to be: temperature 25-29 ± 1 ℃, venting pressure 0.04-0.08Mpa, mixing speed 120-160 rev/min, frequency indication 20.0-26.6 at the condition of culture of fermentation stage.Time 5-7 days.
Technique effect:
The cultural method of mushroom liquid bacterial of the present invention, in common liquid fungus seed culture technology, increased the transition incubation of wood chip pedigree seed culture medium, be that step 2 in the said method does not have in the prior art, this step is because wood chip itself has tiny gap, mushroom mycelium is fully smashed when inoculation cultivate a period of time in this medium after again, just can increase a large amount of mycelia fragments, form a large amount of bacterium points of sending out in the liquid medium within, these bacterium points just can form a large amount of tiny and uniform effective mycelium pellets in proper culture medium.Thereby can turn out the evenly moderate bacterial classification of size and shorten incubation time, from the comparison diagram of accompanying drawing, can see, by forming a large amount of tiny effective bacterium balls behind the bacterial classification inoculation of wood chip pedigree seed culture medium cultivation, diameter reaches more than 90% at the effective bacterium ball between the 0-2mm, has vigor most.And the bacterium ball of the bacterial classification inoculation of directly cultivating by mother culture media is not of uniform size, and big bacterium ball loses vigor because diameter is too big.
The wood chip pedigree seed culture medium is a kind of medium that adds wood chip, cotton seed hull, bean powder and land plaster, and the slight gap of wood chip and cotton seed hull helps mycelial growth, and the adding of other nutrient components such as bean powder can make mycelial growth vigorous.
Special-purpose mother culture media is owing to be a kind of special culture media of cultivating at mushroom liquid bacterial, therefore the leachate that in this medium, has added mushroom waste material culture, wheat bran and the content that has reduced yeast extract have been removed simultaneously, because used hormone substance and contain the holoside nutrient material that is decomposed by primordial hypha but be not absorbed when in the mushroom waste material, containing original mycelia and decomposing the wood chip polysaccharide, so cultivation through special-purpose mother culture media, can shorten incubation time, make mycelial growth vigorous, mycelia is strong.
Submerged fermentation culture medium and original liquid nutrient medium difference, be that it does not contain monose composition (glucose), the required sugared source of bacterium ball growth is from the corn flour and the starch of polysaccharide, the use of these two kinds of materials, not only reduced the cost of medium, also make mycelial growth in more approaching natural environment, corn flour and starch offer the required carbon source of mycelial growth simultaneously.Because this medium has carried out suitable change aspect mineral matter element and agar content, the product of generation just differs widely, so can use different medium according to the tunning of our needs.
Description of drawings:
Accompanying drawing is the bacterial classification of the mycelium inoculation of employing wood chip pedigree seed culture medium cultivation and the comparison diagram of the bacterial classification of the mycelium inoculation that directly adopts mother culture media to cultivate.
Embodiment:
Embodiment 1: the cultural method of mushroom liquid bacterial: present embodiment is a preferred embodiment, and its concrete steps are: (1), mother planted cultivate in mother culture media: get 0.5cm
2Square mushroom is female plants, and is seeded to special mother kind medium culture, and under 23 ± 1 ℃ of conditions, dark culturing 7 days is carried out actication of culture and subculture, forms the female kind of a test tube; The content and the composition of the special-purpose mother culture media preparation of this step are; 100 parts of special-purpose mother culture medias contain 20 parts of potatos, 1.5 parts in mushroom waste material, 2 parts of glucose, 0.05 part of yeast extract, 0.1 part of peptone, KH
2PO
4: 0.1 part, MgSO
47H
2O:0.05 part, 2 parts in agar, water: 76.2 parts is 6.0 by the pH value of the resulting medium of said components; And former potato culture, yeast extract is 0.25 part, also has 5 parts in wheat bran, the special-purpose mother culture media of present embodiment has increased by 1.5 parts in mushroom waste material, has reduced the content of yeast extract, has removed wheat bran; With respect to former potato culture, the incubation time that reaches full bottle can shorten 2 days at least, and mother plants, and mycelial growth is vigorous on special culture media, mycelia is strong.(2), the mycelia culture transferring that will cultivate full packages in mother culture media carries out transition in the wood chip pedigree seed culture medium and cultivates: in the triangular flask by the full bottle mycelia culture transferring to 150 of step (1) gained milliliter, can transplant six bottles, secretly cultivate then, temperature condition is 25 ± 1 ℃, cultivates 15 days.The composition and the weight percent content of wood chip pedigree seed culture medium raw material part are: sucrose 1%, cotton seed hull 72%, wood chip 10%, wheat bran 10%, bean powder 5%, land plaster (CaCO
3) 2%, the wood chip pedigree seed culture medium was mixed by 1: 1.2 with water by above-mentioned raw materials and gets, and its pH value is 6.0; (3) one-level is shaken bottle amplification: the one-level that mycelium inoculation to six that will be above-mentioned six bottles full bottle is 500 milliliters is shaken bottle, to fully the wood chip bacterial classification be smashed in the time of inoculation, make the wood chip bacterial classification form a large amount of mycelia fragments, be increased in the bacterium point in the liquid nutrient medium, these bacterium points are in proper culture medium, just can form a large amount of tiny mycelium pellets, so the bacterial classification of cultivating by the second step sawdust medium shakes in the bottle in one-level, cultivating three days just visible a large amount of even bacterium balls produces, one-level is shaken the condition of culture of bottle: 25 ± 1 ℃ of temperature, 125 rev/mins of frequencies of oscillation, inoculum concentration 10%, the liquid nutrient medium coefficient is 100ml-150ml/500ml (triangular flask), and cultivating fate is 3 days; (4) secondary shakes bottle amplification: one-level is shaken mycelium inoculation to six bottle 5000 milliliters shake in the bottle after the bottle amplification, and it is bottle identical that condition of culture and one-level are shaken; (5) secondary is shaken a bottle bacterium ball that comes out and carry out deep layer row fermentation process, fermentation process comprises seeding tank and two stages of fermentation tank culture: at first secondary is shaken a bottle bacterium ball that comes out and be inoculated in the seeding tank of six 20L, being inoculated into six 200L then contains in the fermentation tank of 80% submerged fermentation culture medium, can produce the mushroom bacterium liquid of 960L, these liquid spawns can loop-carrier heavily be 5 jin about 9600 rods of bacterium rod; The condition of culture of seeding tank and fermentation tank is: 25 ± 1 ℃ of temperature, venting pressure 0.04Mpa-0.06Mpa, 160 rev/mins of mixing speeds, frequency indication 26.6.Above-mentioned one-level is shaken bottle and is submerged fermentation culture medium to the employed liquid nutrient medium of fermentation process, and its composition and weight percent content are: contain 0.2 part of yeast extract in 100 parts of fermentation tank culture medium, 1 part in wheat bran, 2 parts of corn flour, 2 parts of starch, land plaster (CaCO
3) 0.3 part, KH
2PO
4: 0.085 part, MgSO
47H
2O:0.08 part, NaCl:0.02 part, vitamin B1: 0.005 part, 0.1 part in agar, all the other are water.
By the change of liquid nutrient medium, also can cultivate a large amount of bacterium balls, extract the bacterium ball in a large number by filter, under 40-60 ℃ temperature condition, dry, pulverize the back and obtain mushroom bacterium ball dry powder, simultaneously also can the cultured culture fluid of direct marketing.
With the inventive method is the technological process that the basis further produces mushroom, can produce different products (as mushroom strain, bacterium ball dry powder, bacterium rod, mushroom) in the different phase on the same set of equipment, provides feasible foundation for making full use of this complete equipment.
Embodiment 2: the liquid nutrient medium among the embodiment 1 adopts original mushroom liquid nutrient medium, and its composition and content are: contain 20 parts of potatos in 100 fens liquid medium, 2 parts of glucose, (NH
3) SO
4: 0.2 part, KH
2PO
4: 0.1 part, MgSO
47H
2O:0.05 part, vitamin B1: 0.001 part, 0.05 part in agar, all the other are water.Do not contain corn flour and starch in this prescription, but contain potato and glucose (monose), therefore embodiment 1 cost is higher relatively.Other steps of present embodiment and condition are with embodiment 1.
Embodiment 3: the composition of special-purpose mother culture media and content can also for: potato is 15 parts in 100 parts of special-purpose mother culture medias, 3 parts in mushroom waste material, 1 part of glucose, 0.02 part of yeast extract, 0.2 part of peptone, KH
2PO
4: 0.2 part, MgSO
47H
2O:0.07 part, 2 parts in agar, all the other are water.
Embodiment 4: the composition of special-purpose mother culture media and content can also for; Potato is 30 parts in 100 parts of special-purpose mother culture medias, 1.5 parts in mushroom waste material, 1 part of glucose, 0.2 part of yeast extract, 0.05 part of peptone, KH
2PO
4: 0.05 part, MgSO
47H
2O:0.01 part, 1 part in agar, all the other are water.
Embodiment 5: the composition of special-purpose mother culture media and content can also for: potato is 22 parts in 100 parts of special-purpose mother culture medias, 2 parts in mushroom waste material, 3 parts of glucose, 0.1 part of yeast extract, 0.15 part of peptone, KH
2PO
4: 0.1 part, MgSO
47H
2O:0.03 part, 3 parts in agar, all the other are water.
Embodiment 6: the composition of wood chip pedigree seed culture medium raw material part and weight percent content can also be sucrose 0.5%, cotton seed hull 70%, wood chip 5%, wheat bran 10%, bean powder 6%, land plaster (CaCO
3) 2.5%, the wood chip pedigree seed culture medium was mixed by 1: 1.3 with water by above-mentioned raw materials and gets.
Embodiment 7: the composition of wood chip pedigree seed culture medium raw material part and weight percent content can also be sucrose 1.5%, cotton seed hull 75%, wood chip 5%, wheat bran 5%, bean powder 2%, land plaster (CaCO
3) 0.5%, the wood chip pedigree seed culture medium was mixed by 1: 1.4 with water by above-mentioned raw materials and gets.
Embodiment 8: submerged fermentation culture medium, its composition and weight percent content can also for: yeast extract is 0.1 part in 100 fens fermentation tank culture medium, 0.5 part in wheat bran, 5 parts of corn flour, 5 parts of starch, land plaster (CaCO
3) 0.5 part, KH
2PO
4: 0.1 part, MgSO
47H
2O:0.2 part, NaCl:0.1 part, vitamin B1: 0.008 part, 0.9 part in agar, all the other are water.
Embodiment 9: submerged fermentation culture medium, its composition and weight percent content can also for: yeast extract is 0.5 part in 100 parts of medium, 3 parts in wheat bran, 1 part of corn flour, 1 part of starch, land plaster (CaCO
3) 0.05 part, KH
2PO
4: 0.02 part, MgSO
47H
2O:0.01 part, NaCl:0.01 part, vitamin B1: 0.0001 part, 0.05 part in agar, all the other are water.
Claims (9)
1. the submerged culturing method for making of a mushroom liquid bacterial: it is characterized in that comprising the steps: (1), mother planted in mother culture media, cultivate; (2), will in mother culture media, cultivate the mycelia culture transferring that covers with test tube and carry out transition in the wood chip pedigree seed culture medium and cultivate, condition of culture is: temperature 22-27 ± 1 ℃, secretly cultivated 12-20 days, treat that mycelia covers with; (2) the wood chip bacterial classification is fully smashed after immediately culture transferring carry out the cultivation amplification that one-level is shaken bottle in the liquid nutrient medium; (4), one-level being shaken in the bottle 5-7 days bacterium liquid of growth moves into the secondary fill liquid nutrient medium and shakes in the bottle and increase; (5), secondary being shaken the bacterium liquid that comes out of bottle carries out fermentation process and can obtain the finished product mushroom liquid bacterial.
2. the submerged culturing method for making of mushroom liquid bacterial according to claim 1: it is characterized in that described wood chip pedigree seed culture medium is the medium that contains wood chip.
3. the submerged culturing method for making of mushroom liquid bacterial according to claim 1: it is characterized in that described wood chip pedigree seed culture medium is the medium that contains wood chip, cotton seed hull and bean powder.
4. the submerged culturing method for making of mushroom liquid bacterial according to claim 1: it is characterized in that described wood chip pedigree seed culture medium is cultivated carries out in triangular flask.
5. the related wood chip pedigree seed culture medium of the submerged culturing method for making of the described mushroom liquid bacterial of one of claim 1-4, the composition and the weight percent content scope that it is characterized in that this culture medium raw material part are: sucrose 0.5-1.5%, cotton seed hull 70-75%, wood chip 5-10%, wheat bran 5-10%, bean powder 2-6%, land plaster (CaCO
3) 0.5-2.5%, the wood chip pedigree seed culture medium by above-mentioned raw materials and water by 1: 1.2-1.4 mixes and gets, and its pH value is 5.5-6.5
6. the related mother culture media of the submerged culturing method for making of the described mushroom liquid bacterial of one of claim 1-4 is special-purpose mother culture media, it is characterized in that this medium has added mushroom waste material leachate in original potato culture, its pH value scope is 5.5-6.5, this medium component and weight percent content scope are: potato 10-30%, mushroom waste material 1-5%, glucose 1-3%, yeast extract 0.02-2%, peptone 0.05-0.2%, KH
2PO
4: 0.05-0.2%, MgSO
47H
2O:0.01-0.07%, agar 1-3%, water: 60-85%.Condition of culture in the mother culture media is: temperature 20-27 ± 1 ℃, dark culturing, time 5-10 days.
7. the related liquid nutrient medium of the submerged culturing method for making of the described mushroom liquid bacterial of one of claim 1-4 is a submerged fermentation culture medium, this medium component and weight percent content scope are: yeast extract 0.1-0.5%, wheat bran 0.5-3%, corn flour 1-5%, starch 1-5%, land plaster (CaCO
3) 0.05-0.5%, KH
2PO
4: 0.02-0.1%, MgSO
47H
2O:0.01-0.2%, NaCl:0.01-0.1%, vitamin B1: 0.0001-0.008%, agar 0.05-0.9%, water: 86-96%.
8. submerged fermentation culture medium according to claim 7, it is characterized in that adopting the condition of culture of this submerged fermentation culture medium liquid spawn in I and II shakes bottle to be: temperature 25-27 ± 1 ℃, frequency of oscillation 125-155 rev/min, incubation time 3-5 days, inoculum concentration is different according to required product with the liquid nutrient medium coefficient, is respectively 10-15% and 100ml-150ml/500ml.
9. submerged fermentation culture medium according to claim 7, it is characterized in that adopting this submerged fermentation culture medium to be: temperature 25-29 ± 1 ℃ at the condition of culture of fermentation stage, venting pressure 0.04-0.08Mpa, mixing speed 120-160 rev/min, frequency indication 20.0-26.6.Time 5-7 days.
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|---|---|---|---|
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| CN101861797A (en) * | 2010-05-24 | 2010-10-20 | 云南省农业科学院 | Culture method for Lyophyllum Karst liquid spawn |
| CN103168613A (en) * | 2011-12-23 | 2013-06-26 | 何寒 | Fast manufacture method for liquid strain |
| CN103798055A (en) * | 2013-05-26 | 2014-05-21 | 程立君 | Method for producing rhizoma gastrodiae companion armillaria mellea through liquid submerged fermentation |
| CN104106370A (en) * | 2014-06-04 | 2014-10-22 | 保山富群农业科技有限公司 | Liquid strain of edible mushrooms and method for producing liquid strain of edible mushrooms |
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| CN103798055A (en) * | 2013-05-26 | 2014-05-21 | 程立君 | Method for producing rhizoma gastrodiae companion armillaria mellea through liquid submerged fermentation |
| CN104106370B (en) * | 2014-06-04 | 2016-08-24 | 保山富群农业科技有限公司 | A kind of edible fungi liquid strain and production method thereof |
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